The complement (C') system consists of a group of plasma proteins, and cell receptors that play a critical role in non-specific and specific immune response pathways. Despite the fact that C' proteins have been identified in all vertebrates, the evolution of these proteins and their relationships to one another are still unresolved. This laboratory has recently demonstrated, that fish possess multiple isoforms of the third component of C', C3, that are the products of different genes and differ in their function. Furthermore, preliminary experiments indicate that trout possess, in multiple forms, also factor B/C2-like proteins (fB-1 and fB-2), which appear to be involved in the functional diversity of the C' system in these species. The purpose of this project is to study the structure, functions, and aspects of the evolution of the C' system of fish. This proposal consists of three aims: In Aim 1, the structural and phylogenetic relationships of the various trout C3s to each other and to the related C' proteins C4 and C5 will be studied by obtaining and comparing their cDNA and protein sequences. The possible genetic events that have led to the generation of multiple C3 proteins, and C5 will be investigated by analyzing their sequences and studying their genetic linkage and chromosomal localization. Aim 2 investigates the structure and functions of trout C' proteins fB-1 and/or fB-2 proteins and the possibility that they represent ancestral molecules having the activities of both C2 and factor B and analyze the phylogenetic relationship of trout factor B/C2 proteins (fB-1, fB-2) to factor B/C2 from other species. The identity of the factor B/C2 ancestor will be investigated by determining the extent to which the fB-1 and fB-2 proteins function in both the classical and alternative C' pathways, using monoclonal and polyclonal anti-trout fB-1 and fB-2 antibodies and purified proteins in a variety of C' assays. In addition, this aim will include linkage analysis of fB-1 and fB-2 to C3/C4/C5 and to a putative trout MHC class III region, as well as chromosomal localization studies that together with the phylogenetic analysis will enhance our understanding on the evolution of the MHC class III region and also will shed light on the possible genetic events that led to the generation of fB-1 and fB-2 and the mammalian C2 and factor B genes. Aim 3 is an investigation of the biological roles played by the various trout C' proteins and an examination of the possibility that their structural and functional diversities represent a strategy by which these fish have expanded their innate immune system. Binding and affinity of the trout C3, fB, and factor H-like molecule(s) for each other and for bacterial surfaces will be determined in experiments involving purified proteins and whole serum; the interaction(s) of these proteins with infectious hematopoietic necrosis virus (IHNV) and the role played by trout complement in its neutralization will be also investigated. In addition to enhancing our understanding on the evolution of the C' system, the proposed studies are expected to provide new insights into the structure and function of C' proteins and into the mechanism by which C' may be involved in the expansion of the immune recognition capabilities of an animal.